WNV cycles between mosquitoes and birds but also infects humans, horses, and other vertebrate species. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks throughout the United States during the past four years indicate that it has established its presence in the Western Hemisphere. Humans develop a febrile illness that can progress rapidly to a meningitis or encephalitis syndrome (Hubalek et al., 1999, Emerg Inf Dis 5:643-650), and no specific therapy or vaccine has been approved for use in humans.
2.1 Virology
A member of the Flavivirus genus of the Flaviviridae family, WNV is a neurotropic enveloped virus with a single-stranded, positive-polarity 11-kilobase RNA genome. It is translated in the cytoplasm as a polyprotein, and cleaved into structural (C, M, and E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins by virus- and host-encoded proteases. The structural proteins include a capsid protein (C), a transmembrane protein (M) that regulates fusion of the virus with the host membrane, and an envelope protein (E) that functions in receptor binding, membrane fusion, and viral assembly. The role of nonstructural proteins is not fully delineated but these proteins form the viral protease (NS2B, NS3), NTPase (NS3), RNA helicase (NS3), and RNA-dependent RNA polymerase (NS5) (Chambers et al. 1990, Annu. Rev. Microbiol. 44: 649-88). After the E protein of WNV binds to an uncharacterized cell surface receptor, viral uptake is believed to occur through receptor-mediated endocytosis (Chambers et al., 1990, Annu Rev Microbiol 44:649-88). In the endosome, an acid-catalyzed conformational change in E (Gollins et al., 1986, J. Gen. Virol. 67:1941-1950; Kimura et al., 1986, J Gen Virol 67:2423-33) releases the nucleocapsid into the cytoplasm. At the endoplasmic reticulum (ER) membrane, the structural proteins and NS1 undergo co-translational translocation, glycosylation, and membrane-associated cleavage, while the other nonstructural proteins are translated in the cytoplasm (Falgout et al., 1995, J Virol 69:7232-43; Markoffet al., 1994, Virology 204:526-40). Assembly occurs at the ER, and viral particles are exocytosed.
2.2 WNV Immunology
Host factors including immune status influence the expression of WNV disease in humans (Camenga et al., 1974, J Infect Dis 130:634-41). Infants, the elderly, and patients with impaired immune systems are at greatest risk for severe neurological disease (Asnis et al., 2000, Clin Infect Dis 30:413-8; Hubalek et al., 1999, Emerg Inf Dis 5:643-650; Tsai et al., 1998, Lancet 352:767-71). Investigations are beginning to elucidate the molecular basis of WNV infection and the protective immune system response. Maturation of the immune system correlates with resistance to WNV infection (Eldadah et al., 1967, Am J Epidemiol 86:776-90; Eldadah et al., 1967, Am J Epidemiol 86:765-75; Weiner et al., 1970, J Hyg (Lond) 68:435-46). Depletion of macrophages increases the neuro-invasiveness and virulence of an attenuated strain (Ben-Nathan et al., 1996, Arch Virol 141:459-69). Lymphocytes are critical for protection against WNV infection as SCID and RAG1 mice uniformly succumb to infection with WNV (Diamond et al., 2003, J Virol 77:2578-2586; Halevy et al., 1994, Arch Virol 137:355-70). Recent studies demonstrate that components of humoral immunity (IgM, IgG, and complement) have essential functions early in the course of infection and prevent dissemination to the central nervous system (CNS) (Diamond et al., 2003, J Virol 77:2578-2586; Diamond et al., 2003, Viral Immunology 16:259-278). The cellular basis of immunity against WNV is beginning to be delineated. Several studies suggest a protective role for cytotoxic and helper T cells. In vitro, T cells kill targets, proliferate, and release inflammatory cytokines after exposure to WNV-infected cells (Douglas et al., 1994, Immunology 82:561-70; Kesson et al., 1987, J Gen Virol 68:2001-6; Kulkarni et al., 1991, Viral Immunol 4:73-82; Liu et al., 1989, J Gen Virol 70:565-73). In vivo, antigen-specific helper and cytotoxic T cell responses are generated in mice after administration of a candidate vaccine strain of WNV (Yang et al., 2001, J Infect Dis 184:809-16). Although the precise contribution of T cell-mediated immunity in vivo to viral clearance and long-term immunity has yet to be established, recent studies demonstrate an essential role for T cells in the control of WNV infection. Mice that lack CD8+ T cells or classical class I MHC molecules show increased mortality and viral loads, and long-term viral persistence in the CNS after WNV infection (Shrestha et al., 2004, J. Virol. 78:8312-21), and an absence of γδ T cells results in increased mortality after WNV infection (Wang et al., 2003, J Immunol 171:2524-2531).
2.3 Antivirals
At present, treatment for all flavivirus infections, including WNV, is supportive. Ribavirin has been suggested as a candidate agent because it inhibits WNV infection in cells (Jordan et al., 2000, J Infect Dis 182:1214-7); however, its activity was modest at concentrations that are achievable in the CNS (Anderson et al., 2002, Emerg Infect Dis 8:107-8; Jordan et al., 2000, J Infect Dis 182:1214-7). The limited in vivo experience with ribavirin against flaviviruses has not been promising, as it failed to attenuate infection of the closely related Dengue (DEN) virus in mice (Koff et al., 1983, Antimicrob Agents Chemother 24:134-6) and monkeys (Malinoski et al., 1990, Antiviral Res 13:139-49). Based on preliminary cell culture studies (Anderson et al., 2002, Emerg Infect Dis 8:107-8), interferon (IFN) α2b was recently proposed as a possible therapy for WNV. Although in vivo studies have not been performed with WNV, based on experiments with related flaviviruses, IFNs may inhibit WNV dissemination (Harinasuta et al., 1985, Southeast Asian J Trop Med Public Health 16:332-6). Mice that are deficient in IFN α, β, and γ receptors succumb to dengue (DEN) virus infection (Johnson et al., 1999, J Virol 73:783-6) or Murray Valley encephalitis (Lobigs et al., 2003, J Gen Virol 84:567-72) virus infection and mice deficient in IFN γ produced higher viral loads after yellow fever virus infection (Liu et al., 2001, J Virol 75:2107-18). IFN α was effective as prophylaxis and therapy against Saint Louis encephalitis virus in mice (Brooks et al., 1999, Antiviral Res 41:57-64) although clinical benefit was achieved only when therapy was initiated within 24 hours of infection. Indeed, clinical trials on patients with serologically confirmed Japanese encephalitis virus demonstrated no benefit of IFN therapy (Solomon et al., 2003, Lancet 361:821-6). Thus, the window of opportunity for IFN α therapy against WNV infection may be too narrow to be clinically relevant.
The present invention is aimed at addressing the concerns and shortcomings of currents prophylactic and therapeutic methods against flaviviral, particularly WNV, infections.